Simple Binding and Dissociation of a Sialoglycoprotein Using Boronic Acid-Modified Functional Interfaces on Microparticles.

An overexpression of sialic acid is an indicator of metastatic cancer, and selective detection of sialic acid shows potential for cancer diagnosis. Boronic acid is a promising candidate for this purpose because of its ability to specifically bind to sialic acid under acidic conditions. Notably, the binding strength can be easily modulated by adjusting the pH, which allows for a simple dissociation of the bound sialic acid. In this study, we developed 5-boronopicolinic acid (5-BPA)-modified magnetic particles (BMPs) to selectively capture sialic acid biomolecules. We successfully captured fetuin, a well-known sialoglycoprotein, on BMPs at >104 molecules/particle using an acetate buffer (pH 5.0). Facile dissociation then occurred when the system was changed to a pH 7.6 phosphate buffer. This capture-and-release process could be repeated at least five times. Moreover, this system could enrich fetuin by more than 20 times. In summary, BMPs are functional particles for facile purification and concentration through the selective capture of sialic acid proteins and can improve detection sensitivity compared with conventional methods. This technology shows potential for the detection of sialic acid overexpression by biological particles.

Exploration of DPP-IV Inhibitory Peptide Design Rules Assisted by the Deep Learning Pipeline That Identifies the Restriction Enzyme Cutting Site.

The mining of antidiabetic dipeptidyl peptidase IV (DPP-IV) inhibitory peptides (DPP-IV-IPs) is currently a costly and laborious process. Due to the absence of rational peptide design rules, it relies on cumbersome screening of unknown enzyme hydrolysates. Here, we present an enhanced deep learning model called bidirectional encoder representation (BERT)-DPPIV, specifically designed to classify DPP-IV-IPs and explore their design rules to discover potent candidates. The end-to-end model utilizes a fine-tuned BERT architecture to extract structural/functional information from input peptides and accurately identify DPP-IV-Ips from input peptides. Experimental results in the benchmark data set showed BERT-DPPIV yielded state-of-the-art accuracy and MCC of 0.894 and 0.790, surpassing the 0.797 and 0.594 obtained by the sequence-feature model. Furthermore, we leveraged the attention mechanism to uncover that our model could recognize the restriction enzyme cutting site and specific residues that contribute to the inhibition of DPP-IV. Moreover, guided by BERT-DPPIV, proposed design rules for DPP-IV inhibitory tripeptides and pentapeptides were validated, and they can be used to screen potent DPP-IV-IPs.

Improvement of Sensitivity and Speed of Virus Sensing Technologies Using nm- and µm-Scale Components.

Various viral diseases can be widespread and cause severe disruption to global society. Highly sensitive virus detection methods are needed to take effective measures to prevent the spread of viral infection. This required the development of rapid virus detection technology to detect viruses at low concentrations, even in the biological fluid of patients in the early stages of the disease or environmental samples. This review describes an overview of various virus detection technologies and then refers to typical technologies such as beads-based assay, digital assay, and pore-based sensing, which are the three modern approaches to improve the performance of viral sensing in terms of speed and sensitivity.

Hydrogel capsule-based digital quantitative polymerase chain reaction.

Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources.

Levilactobacillus brevis surface layer protein B promotes liposome targeting to antigen-presenting cells in Peyer's patches.

Liposome targeting by conjugation with specific ligands and cross-linking reagents is an attractive strategy for active drug delivery. Here, we demonstrated the potential of surface layer protein (Slp) B from Levilactobacillus brevis JCM 1059 as a specific ligand to antigen-presenting cells (APCs) in Peyer's patches. L. brevis JCM 1059 SlpB-coated liposomes (SlpB-LPs) showed higher resistance to various pH values and bile acids compared to non-coated liposomes (LPs). SlpB-LP showed a significantly higher uptake into dendritic cell-like differentiated THP-1 cells than LP did. The SlpB-LP-conjugated α-galactosylceramide (αGalCer) promoted the production of IL-12 (p40) and TNF-α by THP-1 cells. Furthermore, SlpB-LP showed significantly higher delivery efficiency into APCs underlaying microfold (M) cells in Peyer's patches after oral administration in BALB/c mice and enhanced IL-12 production when αGalCer was conjugated to SlpB-LP. In conclusion, the present study demonstrates the therapeutic potential of SlpB-coated LP to deliver immunomodulatory components to the gut immune system.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC).

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.

Microbial microdroplet culture system (MMC): An integrated platform for automated, high-throughput microbial cultivation and adaptive evolution.

Conventional microbial cell cultivation techniques are typically labor intensive, low throughput, and poorlyparallelized, rendering them inefficient. The development of automated, modular microbial cell micro-cultivation systems, particularly those employing droplet microfluidics, have gained attention for their high-throughput, highly paralellized and efficient cultivation capabilities. Here, we report the development of a microbial microdroplet culture system (MMC), which is an integrated platform for automated, high-throughput cultivation and adaptive evolution of microorganisms. We demonstrated that the MMC yielded both accurate and reproducible results for the manipulation and detection of droplets. The superior performance of MMC for microbial cell cultivation was validated by comparing the growth curves of six microbial strains grown in MMC, conventional shake flasks or well plates. The highest incipient growth rate for all six microbial strains was achieved by using MMC. We also conducted an 18-day process of adaptive evolution of methanol-essential Escherichia coli strain in MMC and obtained two strains exhibiting higher growth rates compared with the parent strain. Our study demonstrates the power of MMC to provide an efficient and reliable approach for automated, high-throughput microbial cultivation and adaptive evolution.

Gel microdroplet-based high-throughput screening for directed evolution of xylanase-producing Pichia pastoris.

Xylanases have useful applications in a wide range of industries. In this regard, Pichia pastoris has become one of the most attractive host platforms for large-scale production of xylanases. However, genomic engineering is still required for overexpression and efficient secretion. In this paper, we applied droplet-based method to screen directed evolved extracellular xylanase producing P. pastoris strain. Xylanase-producing P. pastoris cells were encapsulated in gel microdroplets with a fluorogenic reporter substrate. Improved production of xylanase increases fluorescence intensity of gel microdroplets, enabled accurate selection of evolved clones by droplet sorting. The screening strategy was validated by identifying yeast with improved xylanase production from a mixed sample with a positive selection accuracy of up to 98%. After three rounds of mutagenesis and selection, approximately 108 variants were screened, and a P. pastoris clone with more than 1.3-fold increase in xylanase activity was identified, representing cellular functions improvement of the production host. The throughput of this approach was at least 103-fold higher than that of the robot-assisted microtiter plate reader, and reagent consumption was reduced by ∼106-fold. Furthermore, the greatly shortened incubation time prior screening significantly accelerated the process of directed evolution.

In vivo continuous evolution of metabolic pathways for chemical production.

Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.